De Novo Formula Generation with "Sub-ppm" Confidence using Compass OpenAccess and the micrOTOF

Accurate mass measurements are a key element of chemical characterization. However, the accepted mass accuracy tolerance of 3–5 ppm can still leave significant ambiguity in the proposed chemical formula. Consequently a further input from other analytical techniques such as NMR or MS/MS, along with some judgment based on the synthetic history is often required to arrive at a confident formula assignment.

Traditionally, visual inspection of isotopic information from magnetic sector MS spectra was useful to eliminate spurious formula suggestions. However, to date ESI-TOF technology has been unable to reproduce accurate isotopic patterns and this valuable chemical information has been lost to the characterization task, making automated formula determination almost impossible.

Novel technology embodied in the micrOTOF allows precise measurement of both accurate mass and true isotopic pattern (TIP) over a wide dynamic range, allowing for the implementation of an open access system. This instrumentation enables chemists with limited LC–MS skills to rapidly confirm and identify pharmaceutical compounds with "sub-ppm" confidence in a fully automated workflow.

OpenAccess Software Design

Compass OpenAccess has been implemented with a client server architecture (Figure 1). All information regarding samples, status of the jobs, data, reports and processing results are stored in an Oracle database. The database, located on a central server, serves multiple instruments and computers. Samples can be submitted easily from any PC in the intranet via a web interface. Also the results can be viewed via a simple viewer in the Internet Explorer.

Figure 1.

On the acquisition PC a very simple user interface hides the complexity of the LC–MS system and allows the user to easily queue the submitted samples for automatic acquisition and processing.

Figure 2.

When the sample has been processed, spectra, chromatograms and formula suggestions are sent to the user as a PDF report via e-mail and can also be easily accessed via the web interface (Figure 2 and Figure 3).

Experimental

A set of 113 samples was run with Compass OpenAccess with the goal of unattended formula confirmation. The samples were analysed using an Agilent 1100 liquid chromatography system interfaced with a micrOTOF mass spectrometer under Compass OpenAccess control in a flow injection analysis (FIA) set-up. Lithium formate solution was injected as an external calibration standard.

The data were automatically externally recalibrated using DataAnalysis software. Possible molecular formulae were generated using accurate mass, true isotopic pattern and SigmaFit information with generic atomic search criteria (max. C₂₀₀ H₂₀₀ N₂₀₀ O₂₀₀ Br₃ Cl₃ F₃ S₄, and applying the nitrogen rule). Result filter arguments are SigmaFit < 0.05, ppm tolerance < 5 ppm, maximum 5 formulae suggestion, defined adducts (protonation and sodiated), N2-rule active.

The generated formula assignments were then compared to the recorded formulae.

Results

113 samples were analysed in positive ESI mode using the micrOTOF. Over all average mass error = 1.7 ppm, SD = 4.2 under external calibration.

The sigma interpretation limit was set to 0.05. In mass range from 179 to 821 amu 89% of all results fit into 0.03 sigma limit. All results fit into sigma 0.05.

The relative ppm-error of 3 ppm represents 97% of all results.

ConfirmFormula: For all samples a correct formula result was returned as first suggestion ("top hit"), so the given formula could be automatically confirmed.

FindFormula within limits: For 97% of samples, a correct formula was found as first or second hit suggestion.

Conclusion

De novo formula generation using accurate mass, true isotopic pattern and SigmaFit information allows rapidly confirming and identifying pharmaceutical compounds with "sub-ppm" confidence. This provides a robust base for fully automated formula determination and formula confirmation workflows as implemented in Compass OpenAccess.

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